34 research outputs found
Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50 000 Cells as Starting Material
Histone
post-translational modifications (PTMs) are fundamental
players of chromatin regulation, as they contribute to editing histone
chemical properties and recruiting proteins for gene transcription
and DNA repair. Mass spectrometry (MS)-based proteomics is currently
the most widely adopted strategy for high-throughput quantification
of hundreds of histone PTMs. Samples such as primary tissues, complex
model systems, and biofluids are hard to retrieve in large quantities.
Because of this, it is critical to know whether the amount of sample
available would lead to an exhaustive analysis if subjected to MS.
In this work, we assessed the reproducibility in quantification of
histone PTMs using a wide range of starting material, that is, from
5 000 000 to 50 000 cells. We performed the experiment
using four different cell lines, that is, HeLa, 293T, human embryonic
stem cells (hESCs), and myoblasts, and we quantified a list of 205
histone peptides using ion trap MS and our in-house software. Results
highlighted that the relative abundance of some histone PTMs deviated
as little as just 4% when comparing high starting material with histone
samples extracted from 50 000 cells, for example, H3K9me2 (40%
average abundance). Low abundance PTMs such as H3K4me2 (<3% average
abundance) showed higher variability, but still ∼34%. This
indicates that most PTMs, and especially abundant ones, are quantified
with high precision starting from low cell counts. This study will
help scientists to decide whether specific experiments are feasible
and to plan how much sample should be reserved for histone analysis
using MS
Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications
Identification
of histone post-translational modifications (PTMs)
is challenging for proteomics search engines. Including many histone
PTMs in one search increases the number of candidate peptides dramatically,
leading to low search speed and fewer identified spectra. To evaluate
database search engines on identifying histone PTMs, we present a
method in which one kind of modification is searched each time, for
example, unmodified, individually modified, and multimodified, each
search result is filtered with false discovery rate less than 1%,
and the identifications of multiple search engines are combined to
obtain confident results. We apply this method for eight search engines
on histone data sets. We find that two search engines, pFind and Mascot,
identify most of the confident results at a reasonable speed, so we
recommend using them to identify histone modifications. During the
evaluation, we also find some important aspects for the analysis of
histone modifications. Our evaluation of different search engines
on identifying histone modifications will hopefully help those who
are hoping to enter the histone proteomics field. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange Consortium
with the data set identifier PXD001118
Novel Phosphorylation Sites in the <i>S. cerevisiae</i> Cdc13 Protein Reveal New Targets for Telomere Length Regulation
The <i>S. cerevisiae</i> Cdc13 is a multifunctional
protein
with key roles in regulation of telomerase, telomere end protection,
and conventional telomere replication, all of which are cell cycle-regulated
processes. Given that phosphorylation is a key mechanism for regulating
protein function, we identified sites of phosphorylation using nano
liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS).
We also determined phosphorylation abundance on both wild type (WT)
and a telomerase deficient form of Cdc13, encoded by the <i>cdc13-2</i> allele, in both G1 phase cells, when telomerase is not active, and
G2/M phase cells, when it is. We identified 21 sites of <i>in
vivo</i> phosphorylation, of which only five had been reported
previously. In contrast, phosphorylation of two <i>in vitro</i> targets of the ATM-like Tel1 kinase, S249 and S255, was not detected.
This result helps resolve conflicting data on the importance of phosphorylation
of these residues in telomerase recruitment. Multiple residues showed
differences in their cell cycle pattern of modification. For example,
phosphorylation of S314 was significantly higher in the G2/M compared
to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation
negatively affected telomere length. Our findings provide new targets
in a key telomerase regulatory protein for modulation of telomere
dynamics
Low Resolution Data-Independent Acquisition in an LTQ-Orbitrap Allows for Simplified and Fully Untargeted Analysis of Histone Modifications
Label-free
peptide quantification in liquid chromatography–mass
spectrometry (LC–MS) proteomics analyses is complicated by
the presence of isobaric coeluting peptides, as they generate the
same extracted ion chromatogram corresponding to the sum of their
intensities. Histone proteins are especially prone to this, as they
are heavily modified by post-translational modifications (PTMs). Their
proteolytic digestion leads to a large number of peptides sharing
the same mass, while carrying PTMs on different amino acid residues.
We present an application of MS data-independent acquisition (DIA)
to confidently determine and quantify modified histone peptides. By
introducing the use of low-resolution MS/MS DIA, we demonstrate that
the signals of 111 histone peptides could easily be extracted from
LC–MS runs due to the relatively low sample complexity. By
exploiting an LTQ-Orbitrap mass spectrometer, we parallelized MS and
MS/MS scan events using the Orbitrap and the linear ion trap, respectively,
decreasing the total scan time. This, in combination with large windows
for MS/MS fragmentation (50 <i>m</i>/<i>z</i>)
and multiple full scan events within a DIA duty cycle, led to a MS
scan cycle speed of ∼45 full MS per minute, improving the definition
of extracted LC–MS chromatogram profiles. By using such acquisition
method, we achieved highly comparable results to our optimized acquisition
method for histone peptide analysis (<i>R</i><sup>2</sup> correlation > 0.98), which combines data-dependent acquisition
(DDA)
and targeted MS/MS scans, the latter targeting isobaric peptides.
By using DIA, we could also remine our data set and quantify 16 additional
isobaric peptides commonly not targeted during DDA experiments. Finally,
we demonstrated that by performing the full MS scan in the linear
ion trap, we achieve highly comparable results as when adopting high-resolution
MS scans (<i>R</i><sup>2</sup> correlation 0.97). Taken
together, results confirmed that histone peptide analysis can be performed
using DIA and low-resolution MS with high accuracy and precision of
peptide quantification. Moreover, DIA intrinsically enables data remining
to later identify and quantify isobaric peptides unknown at the time
of the LC–MS experiment. These methods will open up epigenetics
analyses to the proteomics community who do not have routine access
to the newer generation high-resolution MS/MS generating instruments
Novel Phosphorylation Sites in the <i>S. cerevisiae</i> Cdc13 Protein Reveal New Targets for Telomere Length Regulation
The <i>S. cerevisiae</i> Cdc13 is a multifunctional
protein
with key roles in regulation of telomerase, telomere end protection,
and conventional telomere replication, all of which are cell cycle-regulated
processes. Given that phosphorylation is a key mechanism for regulating
protein function, we identified sites of phosphorylation using nano
liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS).
We also determined phosphorylation abundance on both wild type (WT)
and a telomerase deficient form of Cdc13, encoded by the <i>cdc13-2</i> allele, in both G1 phase cells, when telomerase is not active, and
G2/M phase cells, when it is. We identified 21 sites of <i>in
vivo</i> phosphorylation, of which only five had been reported
previously. In contrast, phosphorylation of two <i>in vitro</i> targets of the ATM-like Tel1 kinase, S249 and S255, was not detected.
This result helps resolve conflicting data on the importance of phosphorylation
of these residues in telomerase recruitment. Multiple residues showed
differences in their cell cycle pattern of modification. For example,
phosphorylation of S314 was significantly higher in the G2/M compared
to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation
negatively affected telomere length. Our findings provide new targets
in a key telomerase regulatory protein for modulation of telomere
dynamics
PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization
A novel protein identification framework, PILOT_PROTEIN,
has been
developed to construct a comprehensive list of all unmodified proteins
that are present in a living sample. It uses the peptide identification
results from the PILOT_SEQUEL algorithm to initially determine all
unmodified proteins within the sample. Using a rigorous biclustering
approach that groups incorrect peptide sequences with other homologous
sequences, the number of false positives reported is minimized. A
sequence tag procedure is then incorporated along with the untargeted
PTM identification algorithm, PILOT_PTM, to determine a list of all
modification types and sites for each protein. The unmodified protein
identification algorithm, PILOT_PROTEIN, is compared to the methods
SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both
prepared protein samples and a more complex chromatin digest. The
algorithm demonstrates superior protein identification accuracy with
a lower false positive rate. All materials are freely available to
the scientific community at http://pumpd.princeton.edu
Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane
<div><p>Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is “written” in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.</p></div
Serine/threonine O-acetylation in sugarcane histone H3.
<p>(A) MS/MS spectrum of the [M+2H]<sup>2+</sup> ion (<i>m</i>/<i>z</i> 556.3089) that matched the histone H3 peptide prKSacTGGKprAPR (residues 9–17) where S10 is acetylated. (B) MS/MS spectra of the doubly-charged precursor ion at <i>m</i>/<i>z</i> 598.8534 corresponding to H3T22 acetylation in the H3 peptide prKQLATacKprAAR (residues 18–26). Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.</p
Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.
<p>(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.</p
Lysine methylation and threonine acetylation in sugarcane histone H4.
<p>(A) MS/MS spectrum of the doubly charged ion at <i>m</i>/<i>z</i> 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]<sup>2+</sup> ion at <i>m</i>/<i>z</i> 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.</p